[DNA image-fluorimetry of individual human chromosomes].

Mucrofluorimetric method for the determination of DNA content in individual human chromosomes has been developed. The method is based on a preliminary identification of chromosomes with Hoechst 33258, followed by staining of the chromosomes with Feulgen reaction using Schiffs reagent type ethidium bromide-SO2, then measuring the fluorescence intensity of the chromosomes using an image analyzer. The method allows to determine the DNA content of individual chromosomes with accuracy up to 4.5 fg. DNA content of individual human chromosomes, their p-and q-arms as well as homologous chromosomes were measured using the developed method. It has been shown that the DNA content in the chromosomes of normal human karyotype is unstable. Fluctuations in the DNA content in some chromosomes can vary 35-40 fg.

[Energy corrective and antioxidative actions of cytoflavin during postischemic period of human dermal fibroblasts in vitro].

The influence of metabolic drug Cytoflavin (CF) with antihypoxic and antioxidative properties on human dermal fibroblasts in a model of ischemia-reoxygenation in vitro was studied. It was revealed that the restoration of ATP synthesis in fibroblasts in the postischemic period was considerably accelerated (in 2.1 times) by the addition of CF to the culture medium. The drug had a cell protective effect of reducing cell mortality during the reoxygenation after ischemia by 2-2.7 times. CF effectively reduced the level of reactive oxygen species (ROS) in fibroblasts after H2O2 treatment which allowed maintaining their survival at the level of control cells. Pretreatment of the cells with CF for one day ensured the maintenance of normal levels of ROS during the investigated time period in the fibroblasts subjected to H2O2 treatment, and reduced H2O2-induced cell death by almost a third compared to control cells. The introduction of CF in culture medium after ischemia showed no influence on Hsp70 synthesis, but led to decrease in GRP78 synthesis, raised after ischemia, to the control level, indicating a resolve of the endoplasmic reticulum (ER) stress and functional normalization of ER.

[The cell cycle regulator p130 and beta-catenin form a complex in mesenchymal stem cells].

Suppressor complex p130/E2f4 inhibits transcription of multiple genes proteins regulating cell cycle progression and induces cell cycle arrest at G0/G1 required for induction of cell differentiation in cells of many tissues in vivo and various cell lineages in vitro. We found here that, in mesenchymal stem cells, (MSC) activation of the Wnt/beta-catenin signal pathway induced by MSC coculture with the A-549 cell line or by growth in the medium containing Li+ ions, which resulted in the accumulation of active forms of the p130, E2f4 and beta-catenin, was not coupled with inhibition of cell cycle progression. Cell cycle synchronization of the MSC induced by thymidine and nocodazol was not resulted in change of the levels and phosphorylation pattern of the p130 in contrast to mouse hepatocytes and T98G cells which showed accumulation of the p130 form p1 and p2 in quiescence and form p3 under active proliferative. Antibody to p130 precipitated from extracts of MSC activated by Li+ ions beta the p130 form 2 and hyperphosphorilated beta-catenin. The results obtained suggest that Gsk3beta, p130 and beta-catenin form in MSC a complex the functional role of which may be associated with activation of differentiation not coupled to cell cycle arrest.

[A pocket pRb mutation induces the increase in its affinity to E2F4 coupled with activation of muscle differentiation].

Co-ordination of proliferation and differentiation in cells committed to muscle fate requires the interaction of the retinoblastoma gene product (pRb) with transcription factors of the E2F family. pRb has different affinities for distinct E2Fs, however, the mechanism involved in pRb-E2Fs interaction has not been completely investigated. We have found that pRb carrying a small deletion at the end of the T antigen binding region (deltaS/N), and unable to interact with large T antigen, could induce acute cell cycle block, stable prolongation of the cell cycle in G0/G1 and G2/M phases and suppression of the growth of tumor cells. The deltaS/N showed increased affinity for E2F4, bound hyperphosphorylated forms of E2F4 and induced its nuclear compartmentalization. The ability of deltaS/N to form complexes with E2F4 on DNA was associated with an increase in formation of "free" E2F4 and transsuppression of the specific reporter through preferential binding to E2F4 but not t o E2F1. Stable expression of deltaS/N in multi-potent fibroblasts promoted early muscle commitment. The results obtained suggest that a mutation in the T antigen binding region may increase in affinity of the pRb for E2F4 combined with activation of muscle differentiation.

[Morphological and functional heterogeneity of rat ascitic hepatoma Zajdela cells].

This work is devoted to the study of molecular and cellular mechanisms of disdifferentiation during neoplastic transformation of cells by investigating the malignant tumor cell heterogeneity. We have revealed two cell fractions of hepatoma Zajdela which differ in patterns of growth in primary culture. The cells of one fraction were attached to the culture plastic and grew in a monolayer (S-fraction), whereas cells of another fraction floated in the culture medium (F-fraction). Using method of lifetime supervision of primary culture cells (1-2 passages) at the limit of the resolving power of DIC-microscopy it has been revealed, that both fractions contain cells of several types. Some of them were specific for one of the fractions, and others were found in both fractions, but their frequencies differed. It has been shown by the same method, that long separate cultivation of these fractions in vitro (more than 50 passage) change both cellular structure and the initial ratio of different types of cells in both fractions. According to DNA flow cytometry, the cells of both fractions were hypotetraploid and had insignificant differences in DNA contents. After adaptation to in vitro conditions, S-fraction cells raised their proliferative activity in comparison with the F-fraction cells, and after long cultivation showed 2.3 times higher DNA content. Greater amount of cell surface laminin, a hepatocellular carcinoma marker, was observed on F-fraction cells than on S-fraction cells. Interfractional distinctions were confirmed also by immunologic assessment of hepatoma cells resistance to natural killer lyses: the sensitivity of S-fraction cells in primary culture was 2.4 times higher than F-fraction cells sensitivity, and, after long cultivation, F-fraction cells became practically resistant to cytotoxic action of natural killers. Based on the data obtained, the most probable paths of cell disdifferentiation during hepatoma Zajdela formation and during long cultivation of this tumor cells in vitro are discussed.

[Investigation of the surface heteroorganic antigens of rat hepatoma cells involved in process of cell proliferation].

The investigation of antigenic diversion of hepatoma cells resulting from the expression of heteroorganic kidney antigens has been continued. Tumor-associated heteroorganic antigens 110-115 and 125-130 kDa were detected by immunoserum of narrow specificity in fractions of plasmatic membranes of cells of rat ascitic hepatoma Zajdela and cultured hepatoma HTC; the antigen 75-80 kDa was revealed only for hepatoma Zajdela cells. It has been shown by methods of radioisotope analysis and flow DNA-cytometry that heteroorganic antigens 110-130 kDa can be involved in process of cell proliferation.

[Heat resistance of the skeletal muscle in western palearctic green frogs (Rana esculenta complex)].

Heat resistance of the gastrocnemius muscle was studied in five species of the Rana esculenta complex. It was similar in R. bedriagae, R. lessonae, and in the European form of R. ridibunda; while North African R. saharica demonstrated a lower heat resistance. No heterosis was expressed in R. esculenta, a clonal hybrid of R. lessonae and R. ridibunda, for the heat resistance of the muscle. Moreover, this species demonstrated low heat resistance at the highest test temperatures (42 degrees C). Comparison of diploid and triploid R. esculenta syntopically occurring in the same water bodies demonstrated no heat resistances between them, thus, suggesting that polyploidy has no effect on this parameter at least in this case.

[Variation of microsatellites BM224 and Bcal7 in populations of green toads (Bufo viridis complex) with various nuclear DNA content and ploidy].

We studied variation of microsatellites BM224 and Bcal7 in three species of the Bufo viridis diploid-polyploid complex. We found that locus Bcal7 in all examined samples was monomorphic. Three alleles of microsatellite BM224 were found. Among tetraploid toads, the western species B. oblongus had only one allele variant, whereas the eastern species B. pewzowi had two other alleles. Similar distribution of alleles was observed in triploid specimens, collected in the area borders of tetraploid and diploid species. Among samples of diploid toad B. viridis, we found all three allele variants of microsatellite BM224. Their distribution was geographically determined. A comparison of allele distribution with genome size variation in diploid toads showed very similar patterns.

[Variability in size of the nuclear genome in pygmy wood mouse Sylvaemus uralensis (Rodentia, Muridae)].

Earlier, in an integral genetic study, the Asian and European races were distinguished within the species Sylvaemus uralensis (pygmy wood mouse) and the European race was divided into the East European and South European forms. Each of these groups differed from the others, in particular, in the quantity of the centromeric heterochromatin in karyotypes of the animals. To establish the pattern of its changes in S. uralensis, in the present study the DNA content in splenocyte nuclei in all races and forms of pygmy wood mice was assessed using DNA flow cytometry. The heterochromatin amount in karyotypes and genome size were shown to be correlated. The East European chromosomal race of S. uralensis (Central Chernozem and Non-Chernozem regions of Russia, Crimea Peninsula, Middle Volga region, and Southern Ural) and the Asian race of this species (East Kazakhstan, Uzbekistan, and East Turkmenistan), which have respectively the highest and the lowest amounts of centromeric heterochromatin in the karyotype, exhibit the greatest difference in the DNA content in the genome. On average, the difference is approximately 8% in males and 6.7% in females; in both cases, the ranges of variability were distinctly different. Against the general background of the trait variation, the Asian race, whose members have the smallest DNA amount in their cells, looks homogeneous. The genome of the South European chromosomal form of S. uralensis (Caucasus, Transcaucasia, Carpathians, and Balkan Peninsula), which exhibits an intermediate content of the centromeric heterochromatin in the karyotype, is smaller that the genome of the East European race (by 3.2% in the group of males and by 1.9%, in the group of females), but larger than that of the Asian race (by 5% in either sex). Thus, the variability of size of centromeric C-blocks in pygmy wood mouse is likely to be associated with elimination (or, conversely, an increase in the amount) of the genetically inert chromatin. It is suggested that a significant contribution to the variability of genome size in S. uralensis is made by heterochromosomes, or, more precisely, their variable regions, which seem to be largely heterochromatic.

[Cell shrinkage during apoptosis is not obligatory. Apoptosis of U937 cells induced by staurosporine and etoposide]. / Degidratsionnoe sokrashchenie ob"ema kletok pri apoptoze--fakul'tativnyi priznak. Apoptoz kletok U937, vyzvannyi staurosporinom i étopozidom.

A study was made of apoptotic cell shrinkage, which is generally believed to be a hallmark of apoptosis. The two conventional models of apoptosis were used for examination of changes in cell water balance--one is apoptosis caused in human lymphoma cell line U937 by staurosporine, and the other by etoposide. Intracellular water was determined by measuring buoyant density of cells in continuous Percoll gradient. Apoptosis was recognized by microscopy and flow cytometry. Apoptosis caused by staurosporine (1 microM, 4 h) was found to be associated with a decrease in cell water content by almost 24%. In contrast, no decrease in cell water content was observed in U937 cells incubated with etoposide (50 microM, 4 h), in spite of the number of features suggesting the presence of apoptosis, such as the appearance of apoptotic bodies, chromatin condensation and fragmentation and disappearance of S-phase cells in DNA histogram. It is concluded that definition of apoptosis as "shrinkage-necrosis" (Kerr, 1971) needs correcting: the distinction of apoptotic cells involves the absence of swelling, rather than cell shrinkage.

[High resolution analysis of replication foci by conventional fluorescent microscopy. I. A study of complexity and DNA content of the foci]. / Issledovanie fokusov replikatsii pri vysokom razreshenii metodom shirokopol'noi fluoreschentnoi mikroskopii. I. Analyz kompleksnosti i soderzhaniia DNK v fokusakh.

Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different DNA domains at the functioning replication machinery (functional clustering, model 2). In this study we used conventional fluorescence microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect of hypotonic treatment on overall number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rather arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.

[Effect of narrow fractions of chromatin non-histone proteins on the expression of membrane tumor-associated antigen MA-50 and phosphorylation of proteins of cultured rat hepatocytes]. / Vliianie uzkikh fraktsii negistonovykh belkov khromatina na ékspressiiu membrannogo opukholevoassotsiirovannogo antigena MA-50 i fosfolirovanie belkov kul'tiviruemykh gepatotsitov krysy.

As earlier reported, the main component of narrow fractions of chromosomal non-histone proteins (NHP) of kidney and of Zaidel hepatoma cells has its own protein kinase activity, and is identified as a heteroorgan NHP-antigen, which is intrinsic to the definite renal tissue and absent in the liver. Effects of narrow fractions of kidney and Zaidel hepatoma NHP on biosynthetic processes and sizes of hepatocytes were studied in vitro. It has been shown that as a result of a 5 h incubation of rat hepatocytes with a narrow fraction of renal NHP the proportion of small hepatocytes increases approximately by 12% as compared with that of cells cultivated without NHP. Besides, binding of organ-specific anti-kidney immune serum with a small hepatocyte population rises by more than 20%, which results from the expression of tumor-associated heteroorgan kidney-specific antigen on the hepatocyte surface. According to immunoprecipitation and subsequent electrophoresis, the molecular mass of a membrane heteroorgan antigen on the surface of hepatocytes amounts approximately to 65 kDa, and an active phosphorylation of cellular proteins takes place. The same effect on hepatocytes is produced by a narrow NHP fraction of chromatin of Zaidel hepatoma cells, whereas no phosphorylation is observed in the presence of liver NHP as well as in the absence of NHP. It is suggested that the heteroorgan NHP-antigen induces biosynthetic processes including synthesis of membrane tumorassociated antigen on the surface of hepatocytes cultivated in vitro by activation of cellular protein phosphorylation, which can lead to changes in size of cultivated cells.

[Genetic variation in two forms of the common spadefoot toad Pelobates fuscus (Pelobatidae, Anura, Amphibia) distinguished by genome size]. / Geneticheskaia izmenchivost' u dvukh form obyknovennoi chesnochnitsy Pelobates fuscus (pelobatidae, anura, amphibia), razlichaiushchikhsia po razmeru genoma.

Genetic differences (presumed 23 loci) between two cryptic forms of Pelobates fuscus, differing in genome size, were examined by means of polyacrylamide-gel electrophoresis. This method allowed to discriminate between these forms. Average genetic distance (DN) between both the forms was equal to 0.311, ranging from 0.055 to 0.563. As a rule, differences within these groups were smaller (0.021-0.388). The data show obvious genetic differentiation between these two cryptic forms of P. fuscus. Differences between these forms and P. syriacus were significantly higher (in average 0.943). Genetic distances in relation to speciation and species concepts are discussed.

[Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease]. / Vodnyi i ionnyi balans timotsitov krysy pri apoptoze, vyzvannom deksametazonom i étopozidom. Iono-osmoticheskaia model' umen'sheniia ob'ema kletki.

Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.

[Variability of the level of thermoresistance as an indicator of the functional status of a cell]. / Izmenchivost' urovnia teploustoichivosti kak pokazatel' funktsional'nogo sostoianiia kletki.

Effect of acclimating temperature on the thermoresistance of isolated ciliated gill epithelium of Anodonta anatina was studied. One strip of gills from each mussel was used to determine the initial level of thermoresistance (survival time at 40 degrees C). Other strips were kept at 24 degrees C until their death, and their thermoresistance and fluorescence after Ethidium bromide staining were determined at different time intervals. After a short exposure at 24 degrees C the mean level of thermoresistance of the epithelium increased. The individual shift in the value and direction of cell thermoresistance in different mussels negatively correlated with its initial level. It resulted in narrowing the extent of individual variability of the level of thermoresistance. The prolongation of exposure at 24 degrees C decreased the mean level of thermoresistance, disturbed the negative correlation, and increased the variability of thermoresistance level. The value of cell fluorescence remained constant within 48 h. The increase in fluorescence was observed 12 h after reducing the mean thermoresistance level. Hence, the dynamics of variability in the thermoresistance level is a most sensitive indication of the cell functional state.

[Organization of DNA replication domains in S-phase nuclei of human cells]. / Organizatsiia replikativnykh domenov DNK v S-faznykh iadrakh kletok cheloveka.

Each chromosome of eukaryotic cells contains multiple units of DNA replication that are activated during S-phase of cell cycle according to a definite program. It is considered at present that the main independent units of replication in mammalian cells represent groups of 20-25 adjacent synchronously activated small (with the average size 100 kbp) replicons. After labelling of nascent DNA with nonradioactive DNA precursors and immunofluorescent staining of incorporated label, discrete replication domains (RDs) are detected in S-phase nuclei. It is assumed that each RD is formed by a single group of synchronously activated small replicons. Since the average rate of replication fork movement is 2 kbp/min, a group of small replicons should finish DNA synthesis within 25 min, and only during this time one RD should incorporate the replicative label. We have studied the duration of DNA synthesis in individual RDs in S-phase human cells using double replicative labelling that can be detected in the nucleus by specific reagents. Our results indicate that in the main fraction of RDs DNA synthesis lasts more than 90 min, that contradicts the generally accepted model of organization of replication units in mammalian cells (Hand, 1978), but is in agreement with an alternative model, according to which the main replication units are single or clustered big replicons more than 300 kbp in size (Liapunova, 1994).

[Precise DNA cytometry: investigation of individual variability in animal genome size]. / Pretsizionnaia DNK-tsitometriia: issledovanie individual'noi variabel'nosti razmera genoma zhivotnykh.

The variability of genome size was studied in animal populations and in cell populations of different animal species by means of DNA flow cytometry with the precision level of several tenths of percent. For populations of frog Rana esculenta and laboratory mouse lines C57B1 and CBA the analysis was made with cells of different tissues: erythrocytes for R. esculenta, splenocytes for mice and haploid cells of testes for both species. The results of DNA cytometry, obtained with cells of different species, were shown to be correlated, which indicates the objectivity of individual intrapopulation differences in the genome size recorded with DNA flow cytometry. The level of variability (expressed as CV) was 0.3-0.4% for the population of frogs, and 0.2-0.3% for mouse lines. The analysis of genome size variability in cell populations of different animal species revealed a relationship between the variability level and the genome size: the coefficient of variance of the peak of DNA histogram was inversely related to the square root of genome size. The latter effect may be explained by fluctuations of a measured multicomponent object, but it is still unclear whether these fluctuations could be related to the genome size or to pecularities of chromatin structure. It is concluded that the method of flow DNA cytometry may be effective for studying individual differences in the genome size.

[The heterogeneity of cell populations by the cytochemical characteristics of the cellular chromatin]. / Geterogennost' kletochnykh populiatsii po tsitokhimicheskim kharakteristikam khromatina kletok.

Cell heterogeneity of cytochemical characteristics of chromatin in the norm and after provocative factors in vitro (a heating at 45 degrees C, X-irradiation and joint actions of heating and X-ray irradiation) was investigated in the peripheral blood mononuclear cells of sheep and mouse, the lymphoid cell populations of the murine bone marrow, spleen and thymus. As a criterion of cytochemical heterogeneity were used the distinctions in the rate of staining of cell nuclei with DNA-specific dyes, that was registered by means of measurement of coefficient variation of the DNA-histogram basis peak, with a flow cytometry. Heterogeneity of cytochemical characteristics are dealt with showed a variability of spatial organization of interphase nuclei of single cells. The provocative factors bring about a decrease in the level of the cytochemical characteristics heterogeneity in the sheep mononuclear cells. In the murine cell populations studied no such equalizing of the marker of variability was revealed.

[The morphofunctional characteristics of the deciduous membrane in pregnancy edema]. / Morfofunktsional'nye osobennosti otpadaiushchei obolochki pri otekakh beremennykh.

An analysis of DNA content in decidua cells and decidua cell number in compact decidual layer was performed in 19 women with physiological pregnancy and labour, in 31 women with oedema of pregnancy, in 29 women with light and in 11 women with severe pre-eclampsia. Significant difference in DNA content in the group with oedema of pregnancy and in the groups with other gestosis forms was demonstrated. Thus, a conclusion is made that oedema may have an independent genesis.